Abstract
Autophagy is a cellular self-digestion process activated in response to stresses such as energy deprivation and oxidative stress. However, the mechanisms by which energy deprivation and oxidative stress trigger autophagy remain undefined. Here, we report that activation of AMP-activated protein kinase (AMPK) by mitochondria-derived reactive oxygen species (ROS) is required for autophagy in cultured endothelial cells. AMPK activity, ROS levels, and the markers of autophagy were monitored in confluent bovine aortic endothelial cells (BAEC) treated with the glycolysis blocker 2-deoxy-D-glucose (2-DG). Treatment of BAEC with 2-DG (5 mM) for 24 hours or with low concentrations of H2O2 (100 µM) induced autophagy, including increased conversion of microtubule-associated protein light chain 3 (LC3)-I to LC3-II, accumulation of GFP-tagged LC3 positive intracellular vacuoles, and increased fusion of autophagosomes with lysosomes. 2-DG-treatment also induced AMPK phosphorylation, which was blocked by either co-administration of two potent anti-oxidants (Tempol and N-Acetyl-L-cysteine) or overexpression of superoxide dismutase 1 or catalase in BAEC. Further, 2-DG-induced autophagy in BAEC was blocked by overexpressing catalase or siRNA-mediated knockdown of AMPK. Finally, pretreatment of BAEC with 2-DG increased endothelial cell viability after exposure to hypoxic stress. Thus, AMPK is required for ROS-triggered autophagy in endothelial cells, which increases endothelial cell survival in response to cell stress.
Highlights
Autophagy is a tightly regulated catabolic process involving the degradation of cellular components using lysosomal machinery
We found that reactive oxygen species (ROS), via AMPK activation, increase autophagy by inhibition of mammalian target of rapamycin (mTOR) and Atg4 activity and ROS-triggered autophagy increases endothelial cell survival under hypoxic conditions
Our results demonstrate that 2-DG induces autophagy in endothelial cells, a phenomenon not previously reported in this cell type
Summary
Autophagy is a tightly regulated catabolic process involving the degradation of cellular components using lysosomal machinery. This process plays an important role in cell growth, development, and homeostasis by maintaining a balance between the synthesis, degradation, and subsequent recycling of cellular products. During cellular starvation or nutrient deprivation, increased generation of mitochondrial-derived hydrogen peroxide (H2O2) induces oxidation and consequent inhibition of Atg, the cysteine proteases (autophagins) which play crucial roles in autophagy by proteolytic activation of Atg paralogs for targeting to autophagic vesicles by lipid conjugation, as well as in subsequent deconjugation reactions [16]. 2-DG is reported to activate AMPK, increase ROS in cancer cells [28], and trigger autophagy [29]. We provide the first demonstration that AMPK is required for ROStriggered autophagy in endothelial cells exposed to 2-DG
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